FAT mice reveal that mitochondrial FAT is required to upregulate

Graham P. Holloway, Swati S. Jain, Veronic Bezaire, Xiao Xia Han, Jan F. C. Glatz, Joost J. F. P. Luiken, Mary-Ellen Harper and Arend Bonen

Am J Physiol Regulatory Integrative Comp Physiol 297:960-967, 2009. First published Jul 22, 2009; doi:10.1152/ajpregu.91021.2008 You might find this additional information useful...

This article cites 30 articles, 25 of which you can access free at: http://ajpregu.physiology.org/cgi/content/full/297/4/R960#BIBL This article has been cited by 1 other HighWire hosted article: Cardiac and skeletal muscle fatty acid transport and transporters and triacylglycerol and fatty acid oxidation in lean and Zucker diabetic fatty rats A. Bonen, G. P. Holloway, N. N. Tandon, X.-X. Han, J. McFarlan, J. F. C. Glatz and J. J. F. P. Luiken Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2009; 297 (4): R1202-R1212.

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Am J Physiol Regul Integr Comp Physiol 297: R960–R967, 2009.

First published July 22, 2009; doi:10.1152/ajpregu.91021.2008.

FAT/CD36-null mice reveal that mitochondrial FAT/CD36 is required to upregulate mitochondrial fatty acid oxidation in contracting muscle Graham P. Holloway,1 Swati S. Jain,1 Veronic Bezaire,2 Xiao Xia Han,1 Jan F. C. Glatz,3 Joost J. F. P. Luiken,3 Mary-Ellen Harper,2 and Arend Bonen1 1

Department of Human Health & Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada; 2Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada; and 3 Department of Molecular Genetics, Maastricht University, Maastricht, The Netherlands Submitted 16 December 2008; accepted in final form 15 July 2009

subsarcolemmal; intermyofibrillar; muscle contraction THE REGULATION OF FATTY ACID oxidation at the level of mitochondria has long been attributed to the activity of the malonylCoA (M-CoA) carnitine palmitoyltransferase-I (CPTI) axis (32, 33). However, the IC50 of CPTI for M-CoA is below physiological concentrations of this biological inhibitor (22, 32), and reductions in muscle M-CoA concentrations during exercise in humans cannot account for the observed increase in fatty acid oxidation (24, 25, 28). Collectively, these studies have begun to suggest there are likely additional proteins that

Address for reprint requests and other correspondence: G. Holloway, Human Health & Nutritional Sciences, Univ. of Guelph, Guelph, Canada (e-mail: ghollowa@uoguelph.ca).

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contribute to the regulation of mitochondrial fatty acid oxidation.

Recently, fatty acid translocase (FAT)/CD36, a plasma membrane fatty acid transport protein, has been found on skeletal muscle mitochondrial membranes (4, 10). Administration of sulfo-N-succinimidyl oleate (SSO), a putative specific inhibitor of FAT/CD36, decreased fatty acid oxidation rates (%80%) in isolated mitochondria (4, 10), implicating FAT/ CD36 in the regulation of mitochondrial fatty acid oxidation.

Further support that FAT/CD36 regulates mitochondrial fatty acid oxidation comes from a number of other observations, including the following: 1) FAT/CD36 coimmunoprecipates with CPTI (10, 29, 30), and 2) there is a concomitant increase in mitochondrial fatty acid oxidation and the amount of mitochondrial FAT/CD36 protein in exercising muscle (10, 17), such that mitochondrial FAT/CD36 is positively correlated with mitochondrial fatty acid oxidation rates during exercise (17).

A recent report (18) has challenged the interpretation that FAT/CD36 has a role in mitochondrial fatty acid oxidation, since 1) there were no differences in mitochondrial fatty acid oxidation in wild-type (WT) and FAT/CD36 null (KO) mice, and 2) the reactive ester SSO inhibited mitochondrial fatty acid oxidation to a similar extent in both WT and FAT/CD36 KO mice (18). However, this conclusion may not be warranted, since these authors did find a %25% reduction in mitochondrial fatty acid oxidation in KO mice (18). Failure to detect statistical significance in this 25% reduction appeared to be related to the low statistical power in some aspects of that study (18).

Moreover, the metabolic challenge studies were conducted in mitochondria obtained from resting muscle (18), in which mitochondrial FAT/CD36 content was kept static in WT mice.

This likely limited the differences that could be observed in the respiration rates between WT and KO mice, as we (10, 17) and others (29), have suggested that an increase in mitochondrial FAT/CD36 is essential for upregulating the rates of mitochondrial fatty acid oxidation. Thus, there is controversy as to whether FAT/CD36 is central to increasing mitochondrial fatty oxidation, particularly during muscle contraction.

In the present study, we have examined mitochondrial fatty acid oxidation in relation to FAT/CD36. Specifically, we have 1) determined whether FAT/CD36 KO mice have a reduced rate of palmitate oxidation in isolated subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria, 2) examined the specificity of SSO for FAT/CD36, and 3) determined whether a metabolic challenge (muscle contraction) differentially alters mitochondrial fatty acid oxidation in WT and FAT/CD36 KO animals.

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Holloway GP, Jain SS, Bezaire V, Han XX, Glatz JF, Luiken JJ, Harper ME, Bonen A. FAT/CD36-null mice reveal that mitochondrial FAT/CD36 is required to upregulate mitochondrial fatty acid oxidation in contracting muscle. Am J Physiol Regul Integr Comp Physiol 297: R960 –R967, 2009. First published July 22, 2009; doi:10.1152/ajpregu.91021.2008.—The plasma membrane fatty acid transport protein FAT/CD36 is also present at the mitochondria, where it may contribute to the regulation of fatty acid oxidation, although this has been challenged. Therefore, we have compared enzyme activities and rates of mitochondrial palmitate oxidation in muscles of wild-type (WT) and FAT/CD36 knockout (KO) mice, at rest and after muscle contraction. In WT and KO mice, carnitine palmitoyltransferase-I, citrate synthase, and !-hydroxyacyl-CoA dehydrogenase activities did not differ in subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria of WT and FAT/CD36 KO mice. Basal palmitate oxidation rates were lower (P " 0.05) in KO mice (SS #18%; IMF #13%). Muscle contraction increased fatty acid oxidation ($18%) and mitochondrial FAT/CD36 protein ($16%) in WT IMF but not in WT SS, or in either mitochondrial subpopulation in KO mice. This revealed that the difference in IMF mitochondrial fatty acid oxidation between WT and KO mice can be increased %2.5-fold from 13% under basal conditions to 35% during muscle contraction. The FAT/CD36 inhibitor sulfo-N-succinimidyl oleate (SSO), inhibited palmitate transport across the plasma membrane in WT, but not in KO mice. In contrast, SSO bound to mitochondrial membranes and reduced palmitate oxidation rates to a similar extent in both WT and KO mitochondria (%80%; P " 0.05).

In addition, SSO reduced state III respiration with succinate as a substrate, without altering mitochondrial coupling (P/O ratios). Thus, while SSO inhibits FAT/CD36-mediated palmitate transport at the plasma membrane, SSO has undefined effects on mitochondria. Nevertheless, the KO animals reveal that FAT/CD36 contributes to the regulation of mitochondrial fatty acid oxidation, which is especially important for meeting the increased metabolic demands during muscle contraction.

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palmitate:BSA complex (containing 150 nCi of [1-14C] palmitate, and a final palmitate concentration of 75 )M). The reaction continued for 30 min at 37°C and was terminated with the addition of ice-cold 12 N perchloric acid.

A fraction of the reaction medium was removed and analyzed for isotopic fixation, while gaseous CO2 produced from oxidation of [1-14C] palmitate was measured by acidifying the remaining reaction mixture. Liberated 14CO2 was trapped by benzethonium hydroxide over a 90-min incubation period at room temperature, and the radioactivity was determined.

Inhibition studies were performed by preincubating mitochondria with 200 )M SSO dissolved in DMSO for 15 min. Following the preincubation, mitochondria were washed twice to remove SSO/ DMSO before being resuspended in their original volume. For control purposes the same volume (1 )l) of DMSO (final concentration 0.1%) was added to mitochondria that were not supplemented with SSO. Mitochondrial oxygen consumption measurements. Oxygen consumption was measured in isolated mitochondria (0.2 mg/ml) at 37°C using a Clark-type oxygen electrode (Hansatech, Norfolk, UK) and incubated in standard incubation medium (IM: 120 mM KCl, 1 mM EGTA, 5 mM KH2PO4, 5 mM MgCl2, and 5 mM HEPES; pH 7.4) containing 0.3% defatted BSA and assumed to contain 406 nmol O2/ml at 37°C (27). State III (maximum phosphorylating) respiration was determined with 100 )M ADP, using the following substrates: 10 mM succinate $ 5 )M rotenone, or 40 )M palmitoylcarnitine $ 0.5 mM malate. State IV (nonphosphorylating) respiration was determined following the addition of oligomycin (12 )g/mg protein).

Submitochondrial distribution of SSO. To determine the submitochondrial location of SSO, isolated mitochondria were preincubated for 15 min with radiolabeled SSO (200 )M, 130 nCi [3H]-SSO) and washed twice to remove SSO. Thereafter, mitochondria were lysed by repeated freeze thawing, and repelleted using centrifugation (10,000 g * 10 min). The subsequent supernatant fraction was removed, and the radioactivity was determined in both the supernatant fraction (representing SSO within the mitochondrial matrix) and the pellet (representing SSO bound to mitochondrial membranes).

Mitochondrial enzymatic activities. Isolated SS and IMF mitochondria were used to determine the activities of citrate synthase (CS) and !-hydroxyacyl-CoA dehydrogenase (!-HAD). CS activity was assayed spectrophotometrically at 37°C at 412 nm (31), and !-HAD activity was measured at 340 nm (37°C) (3), after lysing the mitochondria with 0.04% Triton X-100 and repeated freeze-thawing. The forward radioisotope assay was used for the determination of CPTI activity, as described by McGarry et al. (22) with minor modifications, as we have previously reported (1, 2). Briefly, the assay was conducted at 37°C and initiated by the addition of mitochondrial standard reaction medium containing 75 )M P-CoA (L-[3H]carnitine Amersham Bioscience, Buckinghamshire, England). The reaction was stopped after 6 min with the addition of ice-cold HCl. Palmitoyl-[3H] carnitine was extracted in water-saturated butanol in a process involving three washes with distilled water and subsequent recentrifugation steps to separate the butanol phase, in which the radioactivity was counted. Data were normalized to mitochondrial protein.

Muscle contraction and mitochondrial fatty acid oxidation. Rates of mitochondrial fatty acid oxidation and contents of mitochondrial FAT/CD36 were compared in resting and contracting muscles from WT and FAT/CD36 KO mice. Following anesthetization (intraperitoneal injections of pentobarbital sodium, 6 mg/100 g body wt; MTC Pharmaceuticals, Cambridge, ON, Canada), the femoral artery of the control limb was ligated, and the resting hindlimb muscles were excised. For muscle contraction studies, the sciatic nerve of the contralateral limb was exposed, and stimulating electrodes were placed around the nerve. Electrical stimulation was applied for three repetitions of 5 min (train delivery, 100 Hz/3 s at 6 – 8 V; train duration, 200 ms; pulse duration 10 ms), with 2 min of recovery between stimulation bouts. Following stimulation, the hindlimb muscles were excised.

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Animals. FAT/CD36 KO mice were obtained from Dr. Maria Febbraio (Cleveland Clinic, Cleveland OH) (14). Breeding of WT and KO mice was conducted on site at the University of Guelph. Agematched (%8 wk) female WT (n & 40, weighing 22.4 ' 0.8 g) and KO (n & 40, weighing 23.8 ' 0.9 g) mice were used in this study.

Animals were housed in a climate- and temperature-controlled room, on a 12:12-h light-dark cycle, with standard chow and water provided ad libitum. This study was approved by the University of Guelph Animal Care Committee.

Genotyping. Genotyping was confirmed using standard reverse transcription methods as outlined by Qiagen (DNAse easy kit) using the following primer sets; WT forward, 5(-CAG CTC ATA CAT TGC TGT TTA TGC ATG-3(; reverse, 5(-GGT ACA ATC ACA GTG TTT TCT ACG TGG-3(; KO forward, 5(-CAG CTC ATA CAT TGC TGT TTA TGC ATG-3(; and reverse, 5(-CCG CTT CCT CGT GCT TTA CGG TAT C-3( (targeted to the PGKneo cassette).

Giant vesicles and fatty acid transport. Giant vesicles were prepared from murine hindlimb muscles as previously described (8, 9, 20, 21). Briefly, muscle tissues were cut into thin layers (1–3 mm thick) and incubated for 1 h at 34°C in 140 mM KCl-10 mM MOPS (pH 7.4), aprotinin (30 )g/ml), and collagenase type VII (150 U/ml) (Sigma-Aldrich, St. Louis, MO) in a shaking water bath. At the end of the incubation, the supernatant fraction was collected, and the remaining tissue was washed with KCl-MOPS and 10 mM EDTA (SigmaAldrich), which resulted in a second supernatant fraction. Both supernatant fractions were pooled, and Percoll (Sigma-Aldrich), KCl, and aprotinin were added to final concentrations of 3.5% (vol/vol), 28 mM and 10 )g/ml, respectively. The resulting suspension was placed at the bottom of a density gradient consisting of a 3-ml middle layer of 4% Nycodenz (wt/vol) (Sigma-Aldrich) and a 1-ml KCl-MOPS upper layer. This sample was centrifuged at 60 g for 45 min at room temperature. Subsequently, the vesicles were harvested from the interface of the upper and middle layers, diluted in KCl- MOPS, and recentrifuged at 12,000 g for 5 min.

Giant vesicles from WT and KO animals were incubated for 15 min in the presence of 200 )M SSO [dissolved in dimethylsulfoxide (DMSO)], or in the same volume of DMSO (control purposes).

Following this preincubation, vesicles were washed to remove excess SSO/DMSO, and palmitate transport rates were measured as described above (11–13). Briefly, 40 )l of 0.1% BSA in KCl-MOPS, containing unlabeled (15 )M) and radiolabeled 0.3 )Ci [3H]-palmitate, and 0.06 )Ci [14C]-mannitol, were added to 40 )l of vesicle suspension. The incubation was carried out for 15 s. Palmitate uptake was terminated by the addition of 1.4 ml of ice-cold KCl-MOPS, 2.5 mM HgCl2, and 0.1% BSA. The sample was then quickly centrifuged, and the supernatant fraction was discarded. Thereafter, radioactivity was determined in the remaining pellet. Nonspecific uptake was measured by adding the stop solution before the addition of the radiolabeled palmitate solution.

Isolation of mitochondria from skeletal muscle. Differential centrifugation was used to obtain both SS and IMF mitochondrial fractions (2, 5, 10) from hindlimb muscles. All procedures were identical to those previously published by our group (4, 10, 17). Briefly, muscle was homogenized and centrifuged at 800 g for 10 min to separate the SS and IMF mitochondria. The IMF mitochondria were treated with a protease (Subtilisin A, 0.25 )g protease/mg starting wet weight muscle) (Sigma-Aldrich) for exactly 5 min to digest the myofibrils.

Further centrifugation was used to remove the myofibrils, and mitochondria were finally recovered by centrifuging twice at 10,000 g for 10 min.

Mitochondrial palmitate oxidation. Labeled CO2 production from palmitate oxidation and acid-soluble trapped 14C was measured in a sealed system, as we have previously described (4, 17). Briefly, mitochondria (100 )l) were added to a system containing a pregassed modified Krebs Ringer buffer, and the reaction was initiated by a 6:1

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Because of tissue limitations, muscles from two animals were pooled prior to isolating mitochondria.

Western blot analysis. Three WT and KO animals were used to confirm contraction-induced increases in FAT/CD36. These samples were analyzed for total protein (BCA protein assay), and 5 )g of denatured isolated mitochondrial protein were used for Western blot analysis. All samples were separated by electrophoresis on SDSpolyacrylamide gels and transferred to polyvinylidene difluoride membranes. Commercially available antibodies were used to detect cytochrome c oxidase complex IV (COXIV; Invitrogen, Burlington, ON, Canada) and FAT/CD36 (Santa Cruz Biotechnology, Santa Cruz, CA). Blots were quantified using chemiluminescence and the ChemiGenius 2 Bioimaging system (SynGene, Cambridge, UK).

Statistics. All data are presented as the mean ' SE. Two-way ANOVA was used, and when significance was obtained, a Fisher’s LSD post hoc analysis was employed. Statistical significance was accepted at P " 0.05.

RESULTS

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WT and KO animals were not different in mean age or weight (P + 0.05). Genotyping confirmed that homozygous WT and FAT/CD36 KO mice were used in this study.

Isolated mitochondrial enzymatic activities. Enzymatic activities of mitochondrial CPTI, CS, or !HAD did not differ in WT and KO mice (Fig. 1, A–C). Compared with SS mitochondria, IMF mitochondria displayed higher rates of CPTI (%70%; P " 0.05, Fig. 1A), CS (%35%), and !HAD (%80%) activities (P " 0.05, Fig. 1, B and C, respectively) in both WT and KO mice.

Palmitate oxidation and FAT/CD36 content in isolated mitochondria at rest and after muscle contraction. Under basal conditions, KO animals had lower (P " 0.05) rates of palmitate oxidation in both SS (#18%) and IMF (#13%) mitochondria (Fig. 2). Compared with SS mitochondria, palmitate oxidation rates were higher (P " 0.05) in IMF mitochondria (WT $55%; KO $65%) (Fig. 2).

In a separate group of animals, we compared the effects of muscle contraction on mitochondrial fatty acid oxidation. In these animals, we again observed a lower rate of fatty acid oxidation in control muscle SS and IMF mitochondria of FAT/CD36 KO mice (Fig. 3). Muscle contraction increased palmitate oxidation in IMF, but not in SS mitochondria, of WT mice ($18%). In contrast, muscle contraction failed to alter palmitate oxidation in either SS or IMF mitochondria in KO mice (Fig. 3). Hence, the difference in palmitate oxidation between WT and KO animals was amplified %2.5-fold ($35%) in IMF mitochondria, when presented with a metabolic challenge (i.e., muscle contraction). FAT/CD36 protein was not present in KO animals, and under basal conditions, WT SS mitochondria contained more (P " 0.05) FAT/CD36 than WT IMF mitochondria (Fig. 3). In WT animals, muscle contraction did not alter FAT/CD36 content in SS mitochondria, whereas the amount of FAT/CD36 in IMF mitochondria increased following electrical stimulation ($16%, P " 0.05; Fig. 3).

Effects of SSO on fatty acid transport into giant vesicles and on mitochondrial oxidation. As expected, the basal rate of palmitate transport into giant vesicles was lower in FAT/CD36 KO mice compared with WT animals (#14%, P " 0.05, Fig. 4).

SSO inhibited palmitate transport in WT animals (#33%, P " 0.05), but as expected, this inhibitor had no effect on palmitate transport in FAT/CD36 KO mice (Figs. 4 and 5A).

In contrast, SSO inhibited palmitate oxidation by %80% in

Fig. 1. Enzymatic activities of carnitine palmitoyltransferase I (CPTI) (A), citrate synthase (CS) (B), and !-hydroxyacyl-CoA dehydrogenase (!HAD) (C) in subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria in wild-type (WT) and FAT/CD36 knockout (KO) mice. Data are expressed as the means ' SE; n & 4 for each of CPTI, !-HAD, and CS in SS and IMF mitochondria. †Significantly different (P " 0.05) from SS mitochondria.

SS and IMF mitochondria in both WT and KO mice (P " 0.05, Fig. 5B).

To examine whether SSO altered mitochondrial respiration, oxygen consumption was measured in isolated mitochondria.

SSO reduced state III respiration when both succinate and palmitoylcarnitine were used as substrates (Table 1). However, SSO did not alter P/O ratios with either substrate (Table 1).

Experiments with radiolabeled SSO showed that in both WT and KO mice, only minimal SSO (%1.5%) was present within the mitochondrial matrix (3.9 ' 0.6 nmol/mg mitochondrial 297 • OCTOBER 2009 •

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protein), while the vast quantity of SSO (%98.5%) was bound to mitochondrial membranes (290 ' 24, nmol/mg mitochondrial protein; P " 0.05).

DISCUSSION

The present studies have shown that FAT/CD36 contributes to the regulation of mitochondrial fatty acid oxidation, both at rest and when challenged metabolically. Specifically, we have found the following: 1) basal rates of palmitate oxidation are lower in isolated SS and IMF mitochondria of FAT/CD36 KO

Fig. 4. Fatty acid transport into giant sarcolemmal vesicles under basal conditions in WT and FAT/CD36 KO mice in the absence and presence of SSO. Data are expressed as the means ' SE; n & 4 independent experiments for WT and KO. To obtain sufficient tissue, muscles from two mice were pooled for each independent experiment. SSO concentration, 200 )M. *Significantly different (P " 0.05) from control. †Significantly different (P " 0.05) from WT.

mice, and 2) muscle contraction increased mitochondrial fatty acid oxidation in WT mice, whereas this stimulus failed to upregulate mitochondrial fatty acid oxidation in FAT/CD36 KO mice. As expected, 3) we confirmed that SSO inhibits FAT/CD36-mediated fatty acid transport across the plasma membrane, but 4) in marked contrast, in mitochondria, SSO inhibited mitochondrial fatty acid oxidation and respiration in an unknown manner.

Fig. 3. The effect of muscle contraction on palmitate oxidation rates and FAT/CD36 protein (C) in SS and IMF mitochondria in WT (A) and FAT/CD36 KO (B) mice. Data are expressed as means ' SE. In A and B, n & 5 independent experiments for WT and KO. To obtain sufficient tissue, muscles from two mice were pooled for each independent experiment. *Significantly different (P " 0.05) from control. †Significantly different (P " 0.05) from SS mitochondria.

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Fig. 2. Palmitate oxidation rates in SS and IMF mitochondria in WT and FAT/CD36 KO mice. Data are expressed as the means ' SE; n & 13 independent experiments for WT and KO mice. To obtain sufficient tissue, muscles from two mice were pooled for each independent experiment. *Significantly different (P " 0.05) different from WT. †Significantly different (P " 0.05) from SS mitochondria.

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Basal rates of fatty acid oxidation are reduced in FAT/CD36 KO mice. A key finding in the present study is that basal rates of palmitate oxidation are reduced in SS and IMF mitochondria of FAT/CD36 KO mice. We have confirmed this finding in the current study, as basal rates of palmitate oxidation were also reduced in both SS (#15%) and IMF (#15%) mitochondria in a separate group of animals used for the muscle contraction experiments. These data in muscles of FAT/CD36 mice provide direct evidence that FAT/CD36 participates in the regulation of mitochondrial fatty acid oxidation in this tissue. The reduction in basal mitochondrial palmitate oxidation in FAT/ CD36 KO mice observed in the current study (%15%) was slightly less than that previously reported at the whole muscle level (#26%) (7). This greater difference in whole muscle

Table 1. The effect of SSO on oxygen consumption in isolated mitochondria Substrate

Group

State III, nmol O2 ! mg#1 ! min#1

State IV, nmol O2 ! mg#1 ! min#1

RCR

P/O

40 )M Palmitoylcarnitine $0.5 )M Malate

Control SSO Control SSO

100.5'6.4 41.5'9.5* 281.9'15.3 186.9'18.7*

23.1'1.9 16.5'0.4* 117.5'7.1 140.5'11.4

4.4'0.4 2.5'0.6 2.5'0.2 1.4'0.1*

2.98'0.16 2.69'0.03 1.44'0.11 1.38'0.14

10 )M Succinate $5 )M Rotenone

Values are presented as means ' SE (n & 3 or 4). SSO, sulfo-N-succinimidyl oleate; RCR, respiratory control ratio; P/O, xxx. *Significantly different (P " 0.05) from DMSO control experiment.

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Fig. 5. Relative inhibition (%) of fatty acid transport (A) and mitochondrial fatty acid oxidation (B) induced by SSO in WT and FAT/CD36 KO mice. Data are expressed as means ' SE; n & 4 or 5 independent experiments for WT and KO. To obtain sufficient tissue, muscles from two mice were pooled for each independent experiment. SSO concentration, 200 )M. *Significantly different (P " 0.05) from WT.

oxidation likely reflects the ability of FAT/CD36 to affect both plasma membrane fatty acid transport and mitochondrial fatty acid oxidation rates (see Figs. 2 and 4). The reduction in basal mitochondrial fatty acid oxidation in the current study is also, however, slightly less than previously reported with isolated mitochondria (%25%) (18). The degree of attenuation in fatty acid oxidation in mitochondria isolated from FAT/CD36 KO may reflect sex differences, as only female animals were used in the current study, whereas King et al. (18) used male mice.

However, while King et al. (18) concluded that FAT/CD36 is not important in regulating mitochondrial fatty acid oxidation (18), we interpret their results to indicate that there likely was a reduction in mitochondrial fatty acid oxidation in FAT/CD36 KO mice. Specifically, examination of their study (18) indicated that there was a large variance in some of their data.

From this, we calculated that the statistical power was at times quite low and seemed to be insufficient to conclude that mitochondrial fatty acid oxidation is not impaired in FAT/ CD36 KO mice. We have been careful to ensure that statistical power in our oxidation data was sufficiently robust to detect differences in fatty acid oxidation between WT and FAT/CD36 KO mice. Taken altogether, we conclude that the small reductions in the basal rates of mitochondrial fatty acid oxidation in FAT/CD36 KO mice in the present study, and likely in the study by King et al. (18), suggest that mitochondrial FAT/ CD36 has perhaps a limited role in regulating fatty acid oxidation rates under basal conditions.

Because the basal rates of plasma membrane palmitate transport and mitochondrial oxidation are both reduced by %15% in FAT/CD36 KO animals, it would appear that FAT/ CD36 has only a minor role in regulating these processes under basal conditions. Compensatory upregulation by other proteins may be counteracting the effect of ablating FAT/CD36. In the current study, we have not observed changes in the activities of selected enzymes involved in fatty acid oxidation, namely CPTI, CS, or !HAD. However, it is possible that compensatory changes in fatty acid transport protein 1 (FATP1) minimized the effect of ablating FAT/CD36. We have previously shown that both FATP1 and FATP4 are increased in the soleus muscle of these animals (7), which may mask, in part, the contribution of FAT/CD36 to basal plasma membrane fatty acid transport. Similar logic may hold true at the mitochondria, as we have detected a %30% increase in FATP1 in both SS and IMF mitochondria isolated from FAT/CD36 KO animals (Holloway GP and Bonen A, unpublished data). It is currently not known what role FATP1 may play in regulating mitochondrial fatty acid oxidation in mature mammalian muscle. We have previously shown that acute overexpression of FATP1 can increase whole muscle fatty acid oxidation rates (23); however, it is unclear whether this occurs exclusively in response to an

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skeletal muscle suggested a role for FAT/CD36 in regulating mitochondrial fatty acid oxidation. Essential to this logic was the notion that SSO was specific to FAT/CD36. In the current study, we have shown that SSO does not affect plasma membrane fatty acid transport in FAT/CD36 KO animals, indicating SSO specifically blocks FAT/CD36 at the plasma membrane.

This confirms a previous study in the hearts of FAT/CD36 KO mice (15) and other reports showing that 3H-SSO specifically binds to FAT/CD36 at the plasma membrane (26). Despite this, we have confirmed a recent report (18), which found that SSO reduces palmitolycarnitine oxidation rates similarly in mitochondria isolated from either WT or FAT/CD36 KO mice. We have also observed that SSO alters mitochondrial respiration rates without altering P/O ratios. These data suggest that SSO does not uncouple the mitochondria (i.e., unaltered P/O ratios, with either palmitoylcarnitine or succinate). While it is unclear how SSO is affecting mitochondria, the current data showing that 1) SSO binds to mitochondrial membranes and 2) reduces state III respiration with succinate suggest that the inhibitory effect of SSO may be occurring at the level of the electron transport chain.

While the present study suggests that FAT/CD36 participates in mitochondrial fatty acid oxidation, the modest reduction observed in the KO animals suggests it is likely not the only regulator of overall mitochondrial fatty acid oxidation, particularly as CPTI activity is seen to be unaltered. A number of other observations also support this proposition, as 1) the content of FAT/CD36 under basal conditions is higher in the SS mitochondria and the fatty acid oxidation rates in SS mitochondria are lower; 2) with aerobic training in rodents there is a disproportionate increase in SS mitochondrial palmitate oxidation (twofold) rates compared with changes in CPTI activity (%50%) (19), while in IMF mitochondria, fatty acid oxidation rates increased without alterations in CPTI activity (19); 3) in some of our other work, mitochondrial CPTI activity and FAT/CD36 combined, but not independently, provided a strong prediction of mitochondrial fatty acid oxidation [r & 0.90, multiple regression, (4)], and 4) an increase in FAT/ CD36 that coimmunoprecipitated with CPTI was strongly correlated with the concurrent increase in whole body fat oxidation (r & 0.93) (29). Lastly, 5) FATP1 in L6E9 myotubes has been shown to have a collaborative effect with CPTI in regulating fatty acid oxidation rates (30). Collectively, these foregoing observations, in agreement with results from the present study, suggest strongly that a complex of proteins at the mitochondrial membrane appears to regulate mitochondrial fatty acid oxidation. These proteins remain to be fully identified.

Perspectives and Significance The current study provides evidence that FAT/CD36 participates in regulating fatty acid oxidation at the level of mitochondria. We propose that the mobilization of FAT/ CD36 from an intracellular depot to the mitochondrial membrane may be particularly important for increasing mitochondrial fatty acid oxidation during exercise. Additionally, we have previously suggested that FAT/CD36 is located distal to CPTI as a direct result of the observation that SSO inhibited palmitoylcarnitine oxidation without altering CPTI activity (4, 17). However, if SSO directly 297 • OCTOBER 2009 •

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increase in plasma membrane fatty acid transport, or whether FATP1 also contributes at the level of the mitochondria in a similar fashion to FAT/CD36. A recent eloquent series of studies by Sebastian and colleagues (30) has shown that CPTI, FAT/CD36, and FATP1 coimmunoprecipitate, and overexpression of these proteins in L6E9 myotubes increase rates of palmitate oxidation. These data suggest that compensatory changes in mitochondrial FATP1 may attenuate the response of ablating mitochondrial FAT/ CD36. Nevertheless, both gain-of-function (30) and loss-offunction (current data) studies have now suggested that FAT/CD36 contributes to mitochondrial fatty acid oxidation.

Muscle contraction fails to increase mitochondrial fatty acid oxidation in FAT/CD36 KO mice. In the present study, we have demonstrated that the contraction-induced upregulation of mitochondrial fatty acid oxidation requires the presence of mitochondrial FAT/CD36, because in the absence of mitochondrial FAT/CD36, there was no increase in contraction-induced fatty acid oxidation. This is fully consistent with the conclusions drawn in our previous studies (10, 17). Consequently, with muscle contraction, the differences between WT and KO IMF mitochondrial palmitate oxidation rates were amplified further ($35% in WT) beyond those observed under basal conditions ($13% in WT).

The present results also parallel differences in a previous report in WT and FAT/CD36 KO mice at the whole muscle level, in which basal palmitate oxidation rates were slightly higher in WT mice ($26%), while AICAR stimulated fatty acid oxidation much more in muscles of WT mice than in FAT/CD36 KO mice (7). Taken altogether, the present study and others (10, 17, 29) strongly support the notion that mitochondrial FAT/CD36 is required to upregulate, at least a portion of, mitochondrial fatty acid oxidation in response to metabolic challenges such as AICAR and muscle contraction. In support of this interpretation, WT IMF mitochondrial FAT/CD36 protein content increased ($16%) proportionately with the increase in WT IMF fatty acid oxidation ($18%), while neither FAT/CD36 protein nor palmitate oxidation rates were altered in WT SS mitochondria.

It is perhaps not surprising that King and colleagues (18) have previously concluded that FAT/CD36 is not important in the regulation of mitochondrial fatty acid oxidation. All of their measurements were obtained under conditions in which mitochondrial FAT/CD36 remained static in WT animals during a metabolic challenge (state III respiration). This presumably limited the magnitude of the differences in fatty acid oxidation that could be achieved between WT and KO animals. However, in our studies (10, 17) and others (29), an increase in fatty acid oxidation has always been associated with a concurrent increase in mitochondrial FAT/CD36. Thus, an increase in mitochondrial FAT/CD36 appears to be central for upregulating mitochondrial fatty acid oxidation, given 1) that there is a positive relationship between mitochondrial FAT/CD36 and mitochondrial fatty acid oxidation during exercise (r & 0.63) (17), and 2) that with muscle contraction mitochondria completely failed to upregulate mitochondrial fatty acid oxidation in FAT/CD36 KO mice (present study).

Inhibition studies with SSO in isolated mitochondria. Previous inhibition studies using SSO in human (4, 17) and rat (10)

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GRANTS This work was funded by the Canadian Institutes of Health Research (to A. Bonen and M.-E. Harper) and the Natural Sciences and Engineering Research Council of Canada (to A. Bonen and M.-E. Harper); the Netherlands Heart Foundation Grant 2002.T049, the Netherlands Organization for Health Research and Development (NWO-ZonMw grant 40 – 00812-98 – 03075), and the European Commission (Integrated Project LSHM-CT-2004 – 005272, Exgenesis). J. Luiken was the recipient of a VIDI-Innovational Research Grant from the Netherlands Organization of Scientific Research (NWO-ZonMw Grant 016.036.305). J. F. C. Glatz is the Netherlands Heart Foundation Professor of Cardiac Metabolism. A. Bonen is the Canada Research Chair in Metabolism and Health.

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affects the electron transport chain as we have currently hypothesized (reduced state III respiration with succinate without altering P/O ratios), in the presence of SSO, oxidation of all substrates would be impaired without compromising CPTI activity. Therefore, the exact location of FAT/ CD36 on mitochondrial membranes must be revisited. We speculate that FAT/CD36 is located on the outer mitochondrial membrane, proximal to CPTI. FAT/CD36 has a large cytoplasmic loop (16), and in this location could increase the rate of substrate delivery to either acyl-CoA synthetase (palmitate) or CPTI (palmitoyl-CoA). This is similar to the speculated role of FAT/CD36 on the plasma membrane, where the increased delivery of fatty acids to the sarcolemma, increases the rate of membrane insertion of fatty acids and ’flip-flop’ across the membrane (reviewed in Ref. 6). Regardless of the exact mechanism of action at the level of the mitochondria, evidence is mounting to suggest that during exercise FAT/CD36 represents a highly regulated protein with the unique capability of increasing both substrate delivery into the muscle cell via sarcolemmal fatty acid transport and mitochondrial fatty acid oxidation in an as yet unknown manner.

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